The vertebrate hypothalamic-pituitary axis (HP) is the main link between the central nervous system and endocrine system. Although several signal pathways and regulatory genes have been implicated in ... [more ▼]

The vertebrate hypothalamic-pituitary axis (HP) is the main link between the central nervous system and endocrine system. Although several signal pathways and regulatory genes have been implicated in adenohypophysis ontogenesis, little is known about hypothalamic-neurohypophysial development or when the HP matures and becomes functional. To identify markers of the HP, we constructed subtractive cDNA libraries between adult zebrafish hypothalamus and pituitary. We identified previously published genes, ESTs and novel zebrafish genes, some of which were predicted by genomic database analysis. We also analyzed expression patterns of these genes and found that several are expressed in the embryonic and larval hypothalamus, neurohypophysis, and/or adenohypophysis. Expression at these stages makes these genes useful markers to study HP maturation and function. [less ▲]

During the last decades, the production endocrine disrupting chemicals reached such levels that they are now spread all over nature. They are known to be very slowly degraded, decreasing the environmental ... [more ▼]

During the last decades, the production endocrine disrupting chemicals reached such levels that they are now spread all over nature. They are known to be very slowly degraded, decreasing the environmental quality and posing ecological risks. Marine mammals inhabiting polluted environments accumulate high levels of these chemicals, so they can be considered good indicators of marine pollution. Thirteen major organochloride pollutants were chosen to make subject of this study: o,p’-DDD; p,p’-DDD; p,p’-DDE; o,p’-DDT; p,p’-DDT; HCB; α-HCH; β-HCH; γ-HCH; δ-HCH; PCB 138; PCB 153 and PCB 180. All these chemicals will have their endocrine disrupting effects characterized individually and collectively by report gene expression assays. The MCF7-ERE cells used in these assays were produced in the Molecular Biology and Genetic Engineering Laboratory of the University of Liège and are originated from a human mammary carcinoma. They carry a gene expressing the synthesis of luciferase and controlled by oestrogen receptors. Then, the thirteen chemicals will be searched in the blubber tissues of porpoises by the use of gas chromatography-mass spectrometry. The blubber samples will also have their endocrine disrupting effect characterized. For the moment, the estrogenic effects of the HCH isomers were put in evidence. The next steps of this work must be soon accomplished. It is expected a great level of these compounds to be found in the samples. On the other hand, their endocrine disrupting effects and how they act in agonist, antagonist and synergist ways are subject that still needs to be cleared. [less ▲]

During the last decades, the production of endocrine disrupting chemicals reached such elevated levels that they are now spread all over the environment. Endocrine disrupting chemicals are known to be ... [more ▼]

During the last decades, the production of endocrine disrupting chemicals reached such elevated levels that they are now spread all over the environment. Endocrine disrupting chemicals are known to be very slowly degraded, decreasing the environmental quality and causing ecological risks. Marine mammals inhabiting polluted environments accumulate high quantities of these chemicals, and are good indicators of marine pollution Thirteen major organochloride pollutants (known from literature to contaminate north sea porpoise) were chosen in this study – o,p’-DDD; p,p’-DDD; p,p’-DDE; o,p’-DDT; p,p’-DDT; HCB; α-HCH; β-HCH; γ-HCH; δ-HCH; PCB 138; PCB 153 and PCB 180. The thirteen chemicals were tested individually and in mixtures by the use of report gene expression assays. The MCF7-ERE cells used in the assays were originated from MCF7 human mammary tumor cells transfected with the ERE-luciferase reporter system. Preliminary results suggest that o,p’-DDD; p,p’-DDD; o,p’-DDT; HCB; β-HCH; δ-HCH; PCB 138 and PCB 180 are able to stimulate the luciferase expression of MCF7-ERE cells when they are present individually or in mixtures. No synergism was observed within mixtures. When MCF7-ERE cells were exposed to the chemicals and 17β-oestradiol simultaneously, p,p’-DDE; α-HCH; PCB 138 and PCB 180 could raise the luciferase expression in comparison to the exposition of 17β-oestradiol alone; on the other hand, o,p’-DDD; p,p’-DDT; HCB; β-HCH; γ-HCH and δ-HCH seem to inhibit the luciferase expression at low doses. In the next step, the pollutants will be searched and quantified in the blubber tissue of porpoises by use of gas chromatography-mass spectrometry. The blubber tissue samples will also have their endocrine disrupting effects characterized. This work is ongoing and It is expected a relevant level of these compounds to be found in the samples. Conversely, their endocrine disrupting effects and how they act in agonist, antagonist ways are subject that still needs to be studied. [less ▲]

Imazalil (IMA) is a widely used imidazole-antifungal pesticide and. therefore. a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ... [more ▼]

Imazalil (IMA) is a widely used imidazole-antifungal pesticide and. therefore. a food contaminant. This compound is also used as a drug (enilconazole). As intestine is the first site of exposure to ingested drugs and pollutants, we have investigated the effects of IMA, at realistic intestinal concentrations, on xenobiotic-metabolizing enzymes and efflux pumps by using Caco-2 cells, as a validated in vitro model of the human intestinal absorptive epithelium. For comparison, other conazole fungicides, i.e. ketoconazole, propiconazole and tebuconazole. were also studied. IMA induced cytochrome P450 (CYP) 1A1 activity to the same extent as benzo(a)pyrene (B(a)P) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), in a dose-and time-dependent manner. Cell-free aryl hydrocarbon receptor (AhR) binding assay and reporter gene assay suggested that IMA is not an AhR-ligand, implying that IMA-mediated induction should involve an AhR-independent pathway. Moreover, IMA strongly inhibited the CYP3A4 activity in 1,25-vitamin D-3-induced Caco-2 cells. The other fungicides had weak or nil effects on CYP activities. Study of the apical efflux pump activities revealed that ketoconazole inhibited both P-glycoprotein (Pgp) and multidrug resistance-associated protein 2 (MRP-2) or breast cancer resistance protein (BCRP), whereas IMA and other fungicides did not. Our results imply that coingestion of IMA-contaminated food and CYP3A4- or CYP1A1-metabolizable drugs or chemicals could lead to drug bioavailability modulation or toxicological interactions, with possible adverse effects for human health. (C) 2008 Elsevier Ireland Ltd. All rights reserved. [less ▲]

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic ... [more ▼]

Aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor mediating the adverse effects of dioxins and polycyclic aromatic hydrocarbons (PAHs). In this study, we investigated the genetic-, time-, dose-, species- and tissue-dependent AhR-mediated agonistic/ antagonistic activities of three food flavonoids: quercetin, chrysin and genistein. To that end, four stably transfected cell lines were used in cell-based luciferase reporter gene assays: three lines were transformed with the ptKLuc vector harbouring four dioxinresponsive elements (DREs) upstream of the thymidine kinase promoter and the luciferase gene (HepG2-Luc, T-47D-Luc and H4IIE-ULg). The fourth is a patented cell line transformed with a different construct: H4IIE DR-CALUX®. Both H4IIE cells were compared for their genetic construction. Human hepatoma (HepG2-Luc) and human breast tumour (T-47D-Luc) cells were compared for tissue-dependent effects. Rat hepatoma (H4IIE-ULg) and human hepatoma (HepG2-Luc) cellswere compared for species-dependent activities.We concluded that quercetin, chrysin and genistein act in a time-, dose-, species- and tissue-specific way. For example, genistein displayed agonistic activities when exposed to rat hepatoma cells during 6h but not after 24 h. Flavonoids displayed agonistic/antagonistic activities in human breast tumour cells, depending on the exposure time, while in human hepatoma cells, only antagonistic activities of flavonoids were measured. In addition, we report, in all the cells, a synergy between an isoflavone and two food contaminants; the 2,3,7,8-tetrachlorodibenzop- dioxin and 3-methylcholanthrene, a PAH. In rat cells, this synergy occurred when cells were exposed to flavonoids and contaminant for 6h, while it was observed in human cells only after 24 h. [less ▲]

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of lowlevel cocktail ... [more ▼]

The illegal use of anabolic substances in the meat producing industry is an ongoing problem due to the continual production of new synthetic compounds and/or the practice of lowlevel cocktail administration to avoid detection by the surveillance schemes of EU member states National Plan surveillance systems. We present a highly sensitive reporter gene assay and sample extraction procedure based on a two step solid phase extraction and high performance liquid chromatography, developed for the detection of glucocorticoid abuse in bovine urine. The assay is capable of detecting compounds with glucocorticoid bioactivity and is extremely sensitive with an EC50 of 0.79 ngmL−1 for dexamethasone. New or unknown compounds with glucocorticoid bioactivity and low-level cocktail mixtures are detectable by this assay. Cross-reactivity data for a range of 11 -hydroxyglucocorticoids has been provided. This assay shows low interference from the 11-keto prohormones and other steroidal hormones. The assay may be suitable for application in other matrices such as hair. In conclusion this screening assay offers advantages over existing analytical techniques. [less ▲]

in Journal of Gravitational Physiology : A Journal of the International Society for Gravitational Physiology (2008), 15

In mammals, the Sox4 gene is involved in development of endocardial crests, the brain, the lung, teeth, gonads and lymphocytes. Recently, Sox4 was shown to control bone mass and mineralization in mice. In ... [more ▼]

In mammals, the Sox4 gene is involved in development of endocardial crests, the brain, the lung, teeth, gonads and lymphocytes. Recently, Sox4 was shown to control bone mass and mineralization in mice. In zebrafish, two homologs for the mammalian Sox4 are present, sox4a and sox4b. Here we investigate the function of the sox4a and sox4b genes in cartilage and bone development in zebrafish. Therefore, we focus our attention on the first bone structures to be formed, the head skeleton and more precisely the pharyngeal cartilage. We show that both genes are expressed in the pharyngeal region, albeit at different time points during development. Double in situ hybridization experiments are used to exactly define the particular tissues where they are expressed. Furthermore, microinjection experiments of antisense oligonucleotides are used to block translation of these specific genes and to define their precise function during cartilage and bone development. [less ▲]

Motivation: Angiogenesis is the process responsible for the growth of new blood vessels from existing ones. It is also associated with the development of cancer, as tumors need to be irrigated by blood ... [more ▼]

Motivation: Angiogenesis is the process responsible for the growth of new blood vessels from existing ones. It is also associated with the development of cancer, as tumors need to be irrigated by blood vessels for growing. New cancer therapies appear that exploit angiogenesis inhibitors, also called angiostatic agents, to asphyxiate and starve the tumors. Better understanding the regulatory mechanisms that control angiogenesis is thus fundamental. Recently, short non-coding RNA molecules, called micro-RNAs, have been discovered that are involved in post- transcriptional regulation of gene expressions. These molecules bind to RNA messengers following the base pairing rules, preventing them from being translated into proteins and/or tagging them for degradation. The main goal of this work is to use computational approaches to identify micro-RNAs involved in angiogenesis. Method: In order to identify genes involved in angiogenesis, bovine endothelial cells were treated by a known angiogenesis inhibitor [1], prolactin 16K, and their gene expression profile was compared to the profile of untreated cells. The genes were then divided into three classes: up-regulated, down-regulated, and unaffected genes. The 3'UTR regions of these genes were then analysed by machine learning techniques. Different approaches were considered. First, we described each gene by a vector of motif counts in their 3'UTR regions and used machine learning techniques to rank the motifs according to their relevance for separating the genes into the different classes. We considered successively motifs corresponding to the seeds of known micro- RNAs and also all possible motifs of a given length. To rank the motifs, we compared ensemble of decision trees and linear support vector machines. Second, we considered an approach called Segment and Combine that was proposed in [2]. Finally, we also carried out an exhaustive search of all motifs of a given length that satisfy some constraints on specificity and coverage with respect to a given gene category. Results: The ability of the different approaches at identifying relevant motifs was first assessed on genes predicted to be the target of some known miRNAs. In this simple setting, most methods were able to identify the micro-RNA seed. The results obtained on the genes regulated by prolactin 16K are also very encouraging. We were able to identify one micro-RNA already known to play a role in angiogenesis and several motifs are predicted by different approaches as very specific of up- or down-regulation by prolactin 16K. Their relationship with known micro-RNAs is certainly worth exploring. Conclusion: Machine learning approaches are promising techniques for the identification of micro-RNA/gene interactions. Future work will concern the application of the same kind of techniques on promoters for the identification of transcription factor binding sites. [less ▲]